RESULTS:

The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP caused by either a lack of degradation or increased production inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr and involved a reduction of the level of c-di-GMP as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa .

Here the influence of metabolizable sugars on the susceptibility of Escherichia coli to β-lactam antibiotics was investigated. Notably monitoring growth and survival of mono- and combination-treated planktonic cultures showed a 1000- to 10 000-fold higher antibacterial efficacy of carbenicillin and cefuroxime in the presence of certain sugars whereas other metabolites had no effect on β-lactam sensitivity. This effect was unrelated to changes in growth rate. Light microscopy and flow cytometry profiling revealed that bacterial filaments formed due to β-lactam-mediated inhibition of cell division rapidly appeared upon β-lactam mono-treatment and remained stable for up to 18 h. The presence of metabolizable sugars in the medium did not change the rate of filamentation but led to lysis of the filaments within a few hours. No lysis occurred in E. coli mutants unable to metabolize the sugars thus establishing sugar metabolism as an important factor influencing the bactericidal outcome of β-lactam treatment. Interestingly the effect of sugar on β-lactam susceptibility was suppressed in a strain unable to synthesize the nutrient stress alarmone (p)ppGpp. Here to the best of our knowledge we demonstrate for the first time a specific and significant increase in β-lactam sensitivity due to sugar metabolism in planktonic exponentially growing bacteria unrelated to general nutrient availability or growth rate. Understanding the mechanisms underlying the nutritional influences on antibiotic sensitivity is likely to reveal new proteins or pathways that can be targeted by novel compounds adding to the list of pharmacodynamic adjuvants that increase the efficiency and lifespan of conventional antibiotics.

Polymorphonuclear neutrophilic leukocytes (PMNs) play a central role in innate immunity where they dominate the response to infections in particular in the cystic fibrosis lung. PMNs are phagocytic cells that produce a wide range of antimicrobial agents aimed at killing invading bacteria. However the opportunistic pathogen Pseudomonas aeruginosa can evade destruction by PMNs and thus cause persistent infections. In this study we show that biofilm cells of P. aeruginosa recognize the presence of attracted PMNs and direct this information to their fellow bacteria through the quorum sensing (QS) signalling system. The bacteria respond to the presence of PMNs by upregulating synthesis of a number of QS-controlled virulence determinants including rhamnolipids all of which are able to cripple and eliminate cells of the host defence. Our in vitro and in vivo analyses support a ‘launch a shield’ model by which rhamnolipids surround the biofilm bacteria and on contact eliminate incoming PMNs. Our data strengthen the view that cross-kingdom communication plays a key role in P. aeruginosa recognition and evasion of the host defence.

Extracellular DNA is one of the major matrix components in Pseudomonas aeruginosa biofilms. It functions as an intercellular connector and plays a role in stabilization of the biofilms. Evidence that DNA release in P. aeruginosa PAO1 biofilms is controlled by the las-rhl and pqs quorum-sensing systems has been previously presented. This paper provides evidence that DNA release in P. aeruginosa PAO1 biofilms is also under iron regulation. Experiments involving cultivation of P. aeruginosa in microtitre trays suggested that pqs expression DNA release and biofilm formation were favoured in media with low iron concentrations (5 μM FeCl3) and decreased with increasing iron concentrations. Experiments involving cultivation of P. aeruginosa in a flow-chamber system suggested that a high level of iron (100 μM FeCl3) in the medium suppressed DNA release structural biofilm development and the development of subpopulations with increased tolerance toward antimicrobial compounds. Experiments with P. aeruginosa strains harbouring fluorescent reporters suggested that expression of the pqs operon was induced in particular subpopulations of the biofilm cells under low-iron conditions (1 μM FeCl3) but repressed in the biofilm cells under high-iron conditions (100 μM FeCl3).

Many opportunistic pathogenic bacteria rely on quorum sensing (QS) circuits as central regulators of virulence expression. In Pseudomonas aeruginosa QS-regulated gene expression contributes to the formation and maintenance of biofilms and their tolerance to conventional antimicrobials and the host innate immune system. Therefore QS is an obvious target for a novel class of antimicrobial drugs which would function to efficiently block reception of the cognate QS signals in vivo and thereby be capable of inducing chemical attenuation of pathogens. As QS is not directly involved in processes essential for growth of the bacteria inhibition of QS does not impose harsh selective pressure for development of resistance as with antibiotics. Numerous chemical libraries of both natural and synthetic origin have been screened and several QS-inhibitory compounds have been identified. In animal pulmonary infection models such inhibitors have proven able to significantly improve clearing of the infecting bacteria and reduce mortality. In addition several enzymes that are able to inactivate the bacterial QS signal molecules have been identified. This inactivation leads to blockage of QS-mediated virulence of plant pathogens in several models.

Quorum sensing (QS) communication systems are thought to afford bacteria with a mechanism to strategically cause disease. One example is Pseudomonas aeruginosa which infects immunocompromised individuals such as cystic fibrosis patients. The authors have previously documented that blockage of the QS systems not only attenuates Ps. aeruginosa but also renders biofilms highly susceptible to treatment with conventional antibiotics. Filamentous fungi produce a battery of secondary metabolites some of which are already in clinical use as antimicrobial drugs. Fungi coexist with bacteria but lack active immune systems so instead rely on chemical defence mechanisms. It was speculated that some of these secondary metabolites could interfere with bacterial QS communication. During a screening of 100 extracts from 50 Penicillium species 33 were found to produce QS inhibitory (QSI) compounds. In two cases patulin and penicillic acid were identified as being biologically active QSI compounds. Their effect on QS-controlled gene expression in Ps. aeruginosa was verified by DNA microarray transcriptomics. Similar to previously investigated QSI compounds patulin was found to enhance biofilm susceptibility to tobramycin treatment. Ps. aeruginosa has developed QS-dependent mechanisms that block development of the oxidative burst in PMN neutrophils. Accordingly when the bacteria were treated with either patulin or penicillic acid the neutrophils became activated. In a mouse pulmonary infection model Ps. aeruginosa was more rapidly cleared from the mice that were treated with patulin compared with the placebo group.

N-Acyl-l-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.

The pathogenesis of Pseudomonas aeruginosa is at least partially attributable to its ability to synthesize and secrete the siderophore pyoverdin and the two zinc metalloproteases elastase and LasA and its ability to form biofilms in which bacterial cells are embedded in an alginate matrix. In the present study a lysophospholipid 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate [also called monopalmitoylphosphatidic acid (MPPA)] which accumulates in inflammatory exudates was shown to inhibit the extracellular accumulation of P. aeruginosa PAO1 alginate elastase LasA protease and the siderophore pyoverdin. MPPA also inhibited biofilm formation. The inhibitory effects of MPPA occur independently of rpoS expression and without affecting the accumulation of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl-L-homoserine lactone and may be due at least in part to the ability of MPPA to bind divalent cations.

Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfp-based reporter technology has been applied for non-destructive single-cell-level detection of quorum sensing in laboratory-based P. aeruginosa biofilms. It is reported that a synthetic halogenated furanone compound which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra is capable of interfering with AHL-mediated quorum sensing in P. aeruginosa. It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis. In addition it reduces the production of important virulence factors indicating a general effect on target genes of the las quorum sensing circuit. The furanone was applied to P. aeruginosa biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does however affect the architecture of the biofilm and enhances the process of bacterial detachment leading to a loss of bacterial biomass from the substratum.

Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces N-acylhomoserine lactones (AHLs) due to an inactivation of the cepR gene. cepR and the cepI AHL synthase gene together constitute the cep quorum-sensing system of B. cepacia. By using a gene replacement method two defined mutants H111-I and H111-R were constructed in which cepI and cepR respectively had been inactivated. These mutants were used to demonstrate that biofilm formation by B. cepacia H111 requires a functional cep quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment but rather controls the maturation of the biofilm. Furthermore it is shown that B. cepacia is capable of swarming motility a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of B. cepacia is quorum-sensing-regulated possibly through the control of biosurfactant production. Complementation of the cepR mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility per se is not essential for biofilm formation on abiotic surfaces.